IV. Translation

1
IV. Translation


2
IV. Translation

• (3) Translation termination
• i. When a ribosome comes to a nonsense
  codon (or stop
• codon, usually one of UAA, UAG and
  UGA), translation
• stops and polypeptide is released from
  ribosome.
• ii. Stop codons do not encode an amino
  acid, so they have no
• corresponding tRNA.
• iii. Termination requires release factors
  (RF1 and RF2) which
• recognize nonsense codon and promote
  the release of the
• polypeptide form the tRNA and the
  ribosome from the
• mRNA. (Fig. 2.35)

3
Termination of translation at a nonsense codon


4
IV. Translation

• 5. Polycistronic mRNA
• ? In bacteria and archaea, the same mRNA can
  encode more
• than one polypeptide. Such mRNAs,
  called polycistronic
• mRNAs, must have more than one TIR to
  allow simultaneous
• translation of more than one sequence
  of the mRNA.
• (1) Even if the two coding regions overlap,
  the two
• polypeptides on an mRNA can be
  translated independently
• by different ribosomes.
• (2) Translational coupling The translation
  of upstream gene is
• required for the translation of the
  gene immediately
• downstream. The secondary structure
  of the RNA blocks
• translation of the second polypeptide
  unless it is disrupted
• by a ribosome translating the first
  coding sequence.

5
Structure of a polycistronic mRNA

• Even if the two coding regions overlap, the two
  polypeptides on
• an mRNA can be translated independently by
  different ribosomes.

6
IV. Translation

• (3) Polar effect on gene expression - Some
  mutations that affect
• the expression of a gene in a
  polycistronic mRNA can have
• secondary effects on the expression of
  downstream gene.
• i. The insertion of an transcription
  terminator prevents the
• transcription of downstream gene.
• ii. The mutation changing a codon to a
  nonsense codon will
• dissociate the ribosome from mRNA,
  then the translation
• of downstream gene that is
  translationally coupled to the
• upstream gene will not translated.
• (4) ?dependent polarity (as shown in
  Fig.2.38)
• A. Normally the rut site is masked by
  ribosome translating the
• mRNA of gene Y.
• B. If translation is blocked in gene Y
  by a mutation that changes
• the codon CAG to UAG, the ?
  factor can cause transcription
• termination before the RNA
  polymerase reach gene Z.
• C. Only the fragment of gene Y protein
  and mRNA are produced
• and even gene Z is not even
  transcribed into mRNA.

7
Model for translational coupling in polycistronic
mRNA
8
Polarity in transcription of a polycistronic mRNA
transcribed from PYZ.
9
V. Regulation of gene expression
10
1. Transcriptional regulation

• (1) Genes whose products regulate the
  expression of other
• genes are called regulatory genes.
  Their products can be
• either activator or repressor.
• (2) The set of genes regulated by the same
  regulatory gene
• product is called a regulon. If a gene
  product regulates its
• own expression, it is said to be
  autoregulated.
• (3) Bacterial genes are often arranged in an
  operon which
• consists of a promoter region, an
  operator region and
• several structure genes. The mRNA of
  bacteria are made on
• a number of genes whose products
  perform related
• functions. This kind of mRNA is called
  polycistronic mRNA.

11
Transcriptional regulation

• (4) There are two general types of
  transcriptional regulation
• i. In negative regulation, a repressor
  binds to an operator and
• turns the operon off by preventing RNA
  polymerase from
• using or access the promoter. An
  operator sequence can
• be close to (up- or downstream), or
  even overlapping the
• promoter.
• ii. In positive regulation, an activator
  binds to the upstream of
• the promoter at an upstream activator
  site (UAS), where it
• can help RNA polymerase bind to the
  promoter or help
• open the promoter after the RNA
  polymerase binds.

12
Two general types of transcriptional
regulation


13
Lac operon

• A. Bacteria respond to rapidly changing
  environments
• B. Examples
• a. Lac operon(?????)of E. coli(????)
• 1. promoter sequence(?????) RNA polymerase
• (RNA???)
• 2. operator sequence(?????) repressor
• protein
• 3. structural genes (????) Z (beta-
• galactosidase ??????), Y (permease
  ???)
• and A (transferase ???)
• b. regulator gene(????) repressor protein
• (?????)

14
The regulation of gene expression of Lac operon
Operon
Operator
Regulatory gene
Promoter
Lactose-utilization genes
DNA
mRNA
Protei repressr
RNA polymerasecannot attach topromoter
Activerepressor
OPERON TURNED OFF (lactose absent)
DNA
RNA polymerasebound to promoter
mRNA
Protein Repressor
Inactiverepressor
Enzymes for lactose utilization
Lactose
OPERON TURNED ON (lactose inactivates repressor)
15
The ß- galactosidase reaction
16
The lac control region
17
Diauxic growth curve of E. coli grown with a
mixture of glucose and lactose
18
The interaction of promoters and CAP proteins in
Lac operon

• A. CAP proteins are involved in positive
  regulation
• a. positive regulation activator(???)
• b. CAP catabolite activator
  proteins(????????)
• c. CAP binding site
• d. cAMP cyclic adenosine monophosphate(??????
  ?)
• e.  CAP/cAMP complex increasing the
  efficiency the
• ability of RNA polymerase binds to
  promoter.
• B. Catabolite repression(?????) enabling E.
• coli to use glucose (???) preferentially for
• energy even in the presence of lactose or
  other
• complex sugar.
• a.  decreasing the level of cAMP
• b.  permease - nonfunctional

19
Positive Control of lac Operon

• Positive control of lac operon by a substance
  sensing lack of glucose that responds by
  activating lac promoter
• The concentration of nucleotide, cyclic-AMP,
  rises as the concentration of glucose drops

20
The phosphoenolpyruvate (PEP)-dependent sugar
phosphotransferase system (PTS)

• Both HPr and IIA are the components of the PTS,
  which is responsible for transporting certain
  sugars, including glucose.

21
Catabolite repression of the lac operon

• Exogenous glucose
• inhibits both cAMP
• synthesis and the
• uptake of other
• sugars, such as
• lactose.
• Components of the
• cascade
• - HPr, the
• phosphotransferase
• (for histidine protein)
• transfers the
• phosphate from
• IIAGlcP to sugar as
• the sugar is transported.

• - IIAGlc protein has two forms
• IIAGlcP activates adenylate
• cyclase to make cAMP.
• IIAGlc inhibits sugar-
• specific permease that
• transport sugar

22
Upstream activator site (UAS)

1. The aCTD (carboxyl terminus of the a
subunits) binds to UP element (UAS),
and aNTD binds to subunit. (A, B)

2. Some promoters lack a -35 sequence and instead
have what is called extended -10 sequence.
This sequence is recognized not bys4 but,
rather by s3. (C)
23
Hypothesis for CAP-cAMP activation of lac
transcription
24
Proposed CAP-cAMP Activation of lac Transcription

• The CAP-cAMP dimer binds to its target site on
  the DNA
• The aCTD (a-carboxy terminal domain) of
  polymerase interacts with a specific site on
  CAP(ARI activation region I)
• Binding is strengthened between promoter and
  polymerase

• (The asubunit N-terminal and C-terminal domains
  (a-NTD and aCTD, respectively) fold
  independently to form two domains that are
  tethered together by a flexible linker.)

25
V. Regulation of gene expression

• 2. Posttranscriptional regulation Gene
  expression can be
• regulated by
• (1) Inhibition of the translation of the
  gene even after mRNA is
• made (translational regulation).
• (2) Degradation of mRNA as soon as it is
  made or before it can
• be translated .
• (3) The protein product may be degraded by
  other protein,
• called protease.
• (4) By feedback inhibition The final
  product inhibits enzyme
• activity of the first reaction in a
  pathway.

26
V. Regulation of gene expression

• 3. Introns and inteins
• (1) some genes have intervening sequence in
  the region of
• DNA encoding a RNA or protein. These
  sequence can move
• from one DNA to another. These
  sequences must be
• spliced out of RNAs and proteins after
  they are made to
• restore the function of RNAs or
  proteins.
• i. The intervening sequences that
  splice themselves out of
• RNA are called introns which are
  much more common in
• eukaryotic cells.
• ii. The intervening sequences that
  splice themselves out of
• protein are called intein.

27
Feedback inhibition regulation
28
VI. Expression vectors

• _at_ The cloning vectors designed to express (made)
  large amounts
• of proteins for biochemical or structural
  analysis.
• Besides the elements of cloning vectors,
  expression vectors
• should have a promoter including operator,
  TIR including ATG,
• SD sequence and termination codon.
• The gene or DNA sequence inserts into cloning
  site must be
• in-frame with ATG.
• For easy purification of expressed protein, some
  affinity tags
• are also include in the vectors.
• (1) Histidine tag DNA sequence encoding
  six histidine amino
• acids
• i. Histidines binds strongly to
  nickel, and so the protein
• contains histidines will bind to a
  column containing nickel.
• ii. Then the bound protein can be
  eluted by washing the
• column with high concentration of
  imidazole, which also
• binds to nickel and so will
  displace the Hist tag.
• (2) Other tag, such as glutathione
  S-transferase (GST) is used
• often.

29
VI. Expression vectors

• Use pET-15b as an example.

30
VI. Expression vectors


31
Transcriptional and tranlational fusions to
express lacZ


32
VII. Some methods for studying gene
expression - Northern blotting

• Buffer (20 X SSC) /1 L, pH 7.0 175.3 g of
  sodium
• chloride 88.2 g 0f sodium citrate

33
Northern Blots

• You have cloned a cDNA
• How actively is the corresponding gene expressed
  in different tissues?
• Find out using a Northern Blot
• Obtain RNA from different tissues
• Run RNA on an denatureing agarose gel (usually
• containing formaldehyde) and blot to membrane
• Hybridize to a labeled cDNA probe
• Northern plot tells abundance of the transcript
• Quantify using densitometer

• Cytoplasmic mRNA isolated from 8 rat tissues
  probed with GPDH (glyceraldehyde-3-phosphate
  dehydrogenase)

34
VII. Some methods for studying gene
expression Reverse transcription
35
VII. Some methods for studying gene
expression - Primer extension

• Start with in vivo transcription, harvest
  cellular RNA containing desired transcript
• Hybridize labeled oligonucleotide 18nt (primer)
• Reverse transcriptase extends the primer to the
  5-end of transcript
• Denature the RNA-DNA hybrid and run the mix on a
  high-resolution DNA gel
• Can estimate transcript concentration also

36
VII. Some methods for studying gene
expression - S1 nuclease mapping

• Use S1 nuclease mapping to locate the ends of
  RNAs
• and to determine the amount of a given RNA in
  cells at
• a given timeLabel a ssDNA probe that can only
• hybridize to transcript of interest
• - Probe must span the sequence start to finish
• - After hybridization, treat with S1 nuclease
  which
• degrades ssDNA and RNA
• - Transcript protects part of the probe from
• degradation
• - Size of protected area can be measured by gel
• electrophoresis
• Amount of probe protected is proportional to
• concentration of transcript, so S1 mapping can
  be
• quantitative

37
S1 Mapping the 5 End
38
Real-Time PCR

• Real-time PCR quantifies the
• amplification of the DNA as it occurs
• As DNA strands separate, forward and reverse
  primers anneal to DNA strand as that in regular
  PCR reaction.
• A fluorescent-tagged oligonucleotide binds to
  part of one DNA strand

39
Fluorescent Tags in Real-Time PCR

• 1. This fluorescent-tagged
• oligonucleotide serves as a reporter
• probe
• Fluorescent tag at 5-end
• Fluorescence quenching tag at 3-end
• 2. With PCR rounds, the 5 tag is
• separated from the 3 tag
• 3. Fluorescence increases with dNTPs
• incorporation into DNA product
• 4. The whole process takes place
• inside a fluorimeter that measure of
• the fluorescence of tag, which is in
• turn is a measure of the progress
• of the PCR reaction (in real time)
• 5. The reaction can be coupled to RT-
• PCR

40
VII. Some methods for studying gene
expression Biochip (Microarray )

41
Run-Off Transcription

• DNA fragment containing gene to transcribe is cut
  with restriction enzyme in middle of
  transcription region
• Transcribe the truncated fragment in vitro using
  labeled nucleotides, as polymerase reaches
  truncation it runs off the end
• Measure length of run-off transcript compared to
  location of restriction site at 3-end of
  truncated gene
• Size of run-off transcript locates transcription
  start site
• Amount of transcript reflects efficiency of
  transcription

42
Nuclear Run-On Transcription

• Isolate nuclei from cells, allow them to extend
  in
• vitro the transcripts already started in vivo
  in a
• technique called run-on transcription
• RNA polymerase that has already initiated
• transcription will run-on or continue to
  elongate
• same RNA chains
• Effective as initiation of new RNA chains in
  isolated
• nuclei does not generally occur, one can be
  fairly
• confident that any transcription observed in
  the
• isolated nuclei is simply a continuation of
• transcription that was already occurring in
  vivo
• Therefore, the transcripts should reveal not only
  
• transcription rates but also give an idea
  about which
• genes are transcribed in vivo.

43
VII. Some methods for studying gene
expression RNA interference (RNAi)

• 1. Also called cosuppression and
  posttrancriptional gene
• silencing (PTGS)
• 2. RNA interference occurs when a cell encounters
  dsRNA from a
• virus, a transposon, or a transgene (or
  experimentally added
• dsRNA).
• 3. This trigger dsRNA Is degraded into 2123-nt
  fragments (siRNA)
• by an RNaseIII-like enzyme, Dicer.
• 4. The double-stranded siRNA, with Dicer and the
  associated
• protein R2D2, constitute a complex (complex
  B).
• 5. Complex B delivers the siRNA to the RISC
  loading complex
• (RLC), which probably separates the two
  strands of the siRNA
• and transfers the guide strand to the
  RNA-induced slicing
• complex (RISC), which includes a protein
  called
• Argonaute2 (Ago2).

44
VII. Some methods for studying gene
expression RNA interference (RNAi)

• 6. The guide strand of the siRNA then base-pairs
  with
• the target mRNA in the active site in the
  PIWI
• domain of Ago2, which an RNase H-like enzyme
  
• also known as slicer.
• 7. Slicer cleaves the target mRNA in the middle
  of the
• region of its base-pairing with siRNA.
• 8. In an ATP-dependent step, the cleaved mRNA is
• ejected from the RISC, which can then accept
  a new
• molecule of mRNA to be degraded.

45
RNA interference (RNAi)
46
RNA interference (RNAi)

• shRNA (siRNA) hRluc (Renilla luciferase)